Bioactive material

ABSTRACT

The present invention relates to a bioactive material and to a method of producing a bioactive material which is suitable for use as an implant or for use as a bone substitute for repairing bone.

The present invention relates to a material and to a method of producinga material which is suitable for use as an implant or for use as a bonesubstitute for repairing bone.

Restoration of skeletal defects or wounds such as femoral neck fracture,spine fusion and lost teeth is a common procedure. For example, over500,000 hip prosthesis implantations, 250,000 spine fusion surgeries,and 500,000 dental implant surgeries are performed annually in theUnited States alone.

Titanium and its alloys, due to their high toughness and excellentbiocompatibility, are widely used in medical implants such as jointprostheses, fracture fixation devices, and dental implants. Othermaterials commonly used in medical and dental implants, include cobaltchrome, polished zirconium, oxinium (zirconium oxide) and stainlesssteel. However, titanium and these other materials demonstrate poorability to bond to bone chemically, and thus osteolysis and subsequentloosening of implants comprising these materials are common.

The performance of an orthopaedic implant can be influenced by thequality of the interface formed between the implant and bone or bonecement. The development of the implant-to-bone (or cement) interfacerelies on a number of factors including surface area, charge,topography, chemistry and contamination of the implant. Theimplant-to-bone interface is the surface of the implant which interfacesor lies adjacent the bone when implanted.

Various techniques are known to modify the implant-to-bone interfacetopography to enhance implant-to-bone integration. These techniquesinclude plasma spraying and electrochemical anodising of theimplant-to-bone interface surface. Problems associated with plasmaspraying and electrochemical anodising include, the formation of animplant-to-bone interface which has low fatigue strength, demonstratespoor adherence to the implant, and suffers from degradation,delamination or cracking during long term implantation.

A commonly used technique for improving tissue ingrowth into orthopaedicimplants is abrasive particle blasting of the implant surface,alternatively known as grit-blasting or sand blasting. This, costefficient process, imparts a micron scale surface structure by blastingabrasive particles on the implant surface. Such roughened surfaces havebeen shown to promote cell attachment and thus improved physicalimplant-to-bone bonding. Furthermore, the increased area of a roughenedsurface means that more cells can attach to the implant-to-boneinterface which also improves implant-to-bone physical bonding. Theimplant having such a modified implant-to-bone interface demonstratesgood osseointegrative properties even in poor quality bone.

However the technique of abrasive particle blasting can causesignificant changes to surface topography by damaging the metal elementson the surface of the implant. The technique of abrasive particleblasting can also cause heterogeneity of the surface chemistry due tothe presence of abrasive particles embedded in the surface of theimplant. The presence of the abrasive particles contaminate the surfaceof the implant and adversely affect the quality of the implant-to-boneinterface. Furthermore, the abrasive particles can detach from thesurface of the implant, leading to increased wear on the bone, implantand implant site.

Additionally, a percentage of the embedded abrasive particles protrudefrom the surface of the implant causing localised micromotion, movementof the implant relative to the implant site, and disruption of tissueingrowth in the surface of the implant. Up to 40% of the surface area ofthe grit blasted implant can become contaminated with abrasive particleswhich can lead to implant-to-bone interface problems, reducedbio-compatibility of the implant and inflammation of the area local tothe implant.

It is the object of the present invention to provide an implant whichhas an enlarged implant-to-bone interface layer with reduced or nocontamination caused by surface embedded abrasive particles. Theimplant-to-bone interface is the surface of the implant which interfacesor lies adjacent the bone when implanted. It is also an object of thepresent invention to provide an implant which has a bioactive, porousand nano-structured surface layer with improved osteoconductive andosteoinductive properties.

Therefore, according to a first aspect of the invention, there isprovided a material suitable as an implant comprising a metal or metalalloy substrate and a primary layer formed on a surface of thesubstrate, said primary layer having a surface area greater than thesurface area of the substrate. Surprisingly it has been found that theprimary layer according to the present invention, having a surface areagreater than the surface area of the substrate, encourages (to a greaterextent) bone to be formed on the surface. Thus increasing bone formationand giving a secure hold on the implant, giving a greater implantsuccess rate both in terms of speed to recover from the implantoperation and overall success of the implant being secured in place.

In use, the surface of the primary layer of the implant interfaces thebone or bone cement. Thus, the primary layer, or more specifically, thesurface of the primary layer provides the implant-to-bone interface. Theincreased surface area of the primary layer means that a larger surfacearea is presented to surrounding cells/cement for increased cell/cementattachment and hence improved integration with the material and thuswith the implant.

According to a second aspect of the invention, there is provided amethod of forming the material of the first aspect, comprising the stepsof providing a metal or metal alloy substrate and forming a primarylayer on a surface of the substrate such that the surface area of theprimary layer is greater than the surface area of the substrate coveredby the primary layer.

Preferably, the substrate comprises a transition metal, a transitionmetal alloy or a transition metal oxide, for example, titanium, TiAlNb,or titanium oxide. Titanium and its alloys, due to their high toughnessand excellent biocompatibility are ideally suited as orthopaedicimplants. Optionally, the substrate may comprise cobalt chrome, polishedzirconium, oxinium (zirconium oxide), stainless steel, tantalum or anycombination of these. The substrate according to the present inventionmay comprise any metal, or metal alloy, or metal oxide or combination ofthese but suitably it would comprise titanium.

Preferably, the step of forming the primary layer on the metal substratecomprises physically altering the surface of the substrate. Physicallyaltering the surface of the substrate roughens the surface of thesubstrate thereby increasing its surface area. The primary or roughlayer promotes cell attachment and thus physical bonding of the implantto bone or to the implant site. The roughened surface presented by theprimary layer provides a surface area significantly larger than thesurface area of the substrate covered by the primary layer.

The step of physically altering the surface of the substrate to form theprimary layer may comprise, for example, machining, sand blasting orgrit blasting, or any combination of these. Preferably, the physicalaltering step comprises grit blasting the surface of the substrate withabrasive particles such as alumina. The primary layer thus formedpresents a roughened, uneven surface texture of peaks, troughs, pits andtrenches which increases the surface area available for cell attachment.

Alternatively, the step of physically altering the substrate maycomprise, for example, a macro or micro physical surface-treatment inwhich a coating of metallic beads is adhered to the surface of thesubstrate. The beads form a 3D porous geometry on the surface of thesubstrate thereby providing a primary layer having a greater surfacearea than the surface of the substrate covered by the coating.Preferably, the primary layer comprises a double or triple layer ofbeads sintered onto the surface of the substrate. Preferably, the beadsare titanium beads and have a mean diameter of 328 μm.

Alternatively, or in addition, the coating may contain a sponge or foamlike network of metallic fibres and/or wires. Alternatively, thesubstrate itself can be porous or sponge like, negating the requirementto physically treat the surface of the substrate. Preferably, the foamor sponge-like structure is composed of sintered beads having diametersof between 15 and 50 μm and pore diameters of several hundred microns toapproximately 1 mm.

In addition, and subsequent to the physically formed primary layer, themethod of forming or completing the primary layer ideally includeschemically treating the physically formed primary layer. The step ofchemically treating the physically formed primary layer comprisessoaking the substrate in an alkaline solution at approximately 30-90° C.The titanium or titanium alloy reacts with the alkaline solution to formalkali titanates. The surface of the completed primary layer thuscomprises alkali titanates. Typically, the surface of the completedprimary layer also includes titanium oxide or titanium oxides.

Preferably, the temperature of the alkaline solution is between 50-70°C. and more preferably between 55-65° C.

It has been found that to heat the substrate or alkaline solution to ahigher temperature can compromise the integrity of the primary layer soformed. For example, where the substrate or alkaline solution is heatedto or above 150° C., a primary layer having a deposit of alkalititanates of a thickness in the micron scale will form. The thicker thealkali titanate deposit or layer, the greater will be the risk ofdelamination or cracking of the alkali titanate layer. Thus the alkalititanate layer, which will in effect form the implant-to-bone interface,bonding the implant to the bone, may be weak and ultimately fail causingseparation of the implant from the bone.

Preferably, the substrate is soaked in an alkaline solution for between1 and 24 hours. Typically, the soaking time is between 1 and 5 hours butis preferably between 1 and 3 hours. It has been found that soakingtimes above 5 hours but in particular above 24 hours also produce aprimary layer having a thickness in the micron scale.

The alkali titanate layer creates a surface to the primary layer whichcomprises a nanostructure of alkali titanates. A nanostructure ornano-textured surface generally means a surface which includes particlesor elements of a size falling within the nanometer range. Thenanostructure of alkali titanates resembles a strut-like morphologycontaining discrete elements, structurally resembling fibres or fibrils,of alkali titanate having a width of between 1 and 20 nanometers (nm).The fibrils are generally cylindrical in shape.

Typically, the length of the fibrils range from 200-300 nm and thedistance between fibrils ranges from 5 nm to 80 nm. The fibrils aregenerally overlaid or stacked one atop another forming the alkalititanate layer or surface. Preferably, the thickness of the alkalititanate layer is in the range of 100-500 nanometers, more preferably100-300 nanometers.

The physical treatment step creates the primary layer having anincreased surface area in preparation for the formation of the alkalititanate nanostructure. The nanostructure of the alkai titanate layercompletes the primary layer and significantly increases the surface areaof the primary layer and hence implant-to-bone interface surface areaavailable for cell attachment and integration. The alkali titanate layeralso masks the adverse affects caused by the presence of any abrasiveparticles present in the implant-to-bone interface of the implant.

Preferably, the primary layer has a surface area of between 1000 and50000 times greater than the surface area of the substrate covered bythe primary layer. More preferably, the primary layer has a surface areaof between 20000 and 50000 times and ideally between 40000 and 50000times greater than the surface area of the substrate covered by theprimary layer.

Typically, the alkaline solution comprises a hydroxide. Preferably, thehydroxide is sodium hydroxide. Other hydroxides can be used with thepresent invention, e.g. lithium hydroxide or potassium hydroxide or anyother suitable metal hydroxide. In this case, the alkali titanatenanostructure of the primary layer will be sodium titanate. Sodiumtitanate is an ionic compound that can be readily modified byion-exchange chemistry into other compounds such as lithium titanate orstrontium titanate to confer different physico-chemical orbiocompatibility characteristics suitable for different applications.The concentration of the hydroxide solution is preferably between 2 and8 molar, more preferably between 3 and 6 molar, and ideally 4 molar.Higher concentrations of hydroxide can lead to re-dissolution of thenanostructure.

The primary layer formed is typically hydrophilic in nature. This isgenerally due to the chemical treatment step in completing the primarylayer. The hydrophilic nature of a material is generally measured by thecontact angle water forms on its surface. The smaller the contact anglethe greater the hydrophilic nature of the material. Preferably, thecontact angle of the primary layer is less than 5°, more preferably isless than 3°.

Preferably, the primary layer has a low reflectance to visible light.Typically, the primary layer has a reflectance to visible light in therange of 1% to 20%. More preferably, the primary layer has a reflectanceto visible light in the range of 5% to 15% and ideally in the range of6% to 10%. The reflectance range gives the primary layer a black colour.

Preferably, the primary layer includes hydroxyapatite, for examplecalcium hydroxyapatite. Typically, the hydroxyapatite is incorporated inthe primary layer by soaking the material in mixed buffer salts.

The material may be used in both medical and dental implants forimproved implant-to-bone integration. More specifically, the materialmay be used in bone replacement implants including, for example, kneejoint, hip joint and shoulder joint prosthesis, femoral neckreplacement, spine replacement and repair, neck bone replacement andrepair, jaw bone repair, fixation and augmentation, transplanted bonefixation, and other limb prosthesis.

Embodiments of the invention will now be described by way of exampleonly and with reference to the accompanying drawings, in which:—

FIG. 1 is a scanning electron micrograph (SEM) of a titanium alloysurface;

FIG. 2 is an SEM of the titanium alloy surface of FIG. 1 after gritblasting with alumina particles;

FIG. 3 is an SEM of a titanium alloy porous beaded surface;

FIG. 4 is an SEM of a titanium alloy sintered bead foam surface;

FIG. 5a is an SEM of titanium alloy surface after grit blasting withalumina particles;

FIGS. 5b-5g are SEMs of samples of the titanium alloy surface of FIG. 5aafter soaking in a 2M (2 molar), 3M, 4M, 6M, 8M and 10M solutionrespectively of sodium hydroxide solution at 60° C. for 2 hours;

FIG. 6a-6c are SEMs of an alumina grit blast titanium alloy surface, atitanium porous beaded surface, and a titanium sintered bead foamsurface respectively, soaked in a 4M sodium hydroxide solution at 60° C.for 2 hours;

FIGS. 7 and 8 are magnified views of the titanium alloy surfaces FIG. 6band FIG. 6c respectively;

FIG. 9 is an SEM of a Porous Beaded Titanium surface prior to formingthe primary layer;

FIG. 10 is an SEM of the porous beaded titanium surface of FIG. 9, theprimary layer having been formed by soaking in a 4M sodium hydroxidesolution at 60° C. for 2 hours in a sonicating water bath;

FIG. 11a is an SEM of the Porous Beaded Titanium surface of FIG. 9soaked in a 2M solution of sodium hydroxide at 60° C. for 10 minutes;

FIG. 11b is a magnified SEM of the Porous Beaded Titanium surface ofFIG. 11a , more clearly showing the early formation of thenanostructured primary layer comprising nano-sized fibrils having a sizein the region of 1-20 nanometers;

FIG. 11e is an SEM of the Porous Beaded Titanium surface of FIG. 11asoaked in a 2 Molar solution of sodium hydroxide at 60° C. for a further15 minutes clearly showing the development of the nanostructured primarylayer;

FIG. 11d is an SEM of a different portion of the Porous Beaded Titaniumsurface of FIG. 11a clearly showing the irregular nature of theformation of the primary layer;

FIG. 12 is an SEM of a commercially pure titanium surface after gritblasting with alumina particles with subsequent soaking in a 4M solutionof sodium hydroxide solution at 60° C. for 2 hours;

FIGS. 13a-13c are SEMs of increasing magnification of areas of thesurface of TiAlNb alloy after grit blasting with alumina particles butprior to soaking in sodium hydroxide, the SEM employing a 2 kv beam toanalyse the upper structure of the primary layer created;

FIGS. 14a-14c are SEMs of the same areas of the surface of the TiAlNballoy of FIGS. 13a-13c after soaking in 4M sodium hydroxide solution at60° C. for 2 hours, the SEM employing a 2 kv beam to analyse the upperstructure of the completed primary layer;

FIGS. 15a and 15b are SEMs of the same areas of the surface of theTiAlNb alloy of FIGS. 14b and 14c respectively, the SEM employing a 15kv beam to analyse the substructure of the completed primary layer;

FIG. 16 is a graph showing the percentage reflectance from the surfaceof the primary layer for different substrates; and

FIGS. 17a and 17b are pictorial views of samples of the materialaccording to the present invention showing the primary layer prior totreatment with sodium hydroxide and post treatment with sodium hydroxiderespectively.

FIG. 18 shows grit blasted titanium coupons p-NPP data normalised to DNA(Pico Green) with error bars of standard deviation. This data is fromTable 2.

FIG. 19 shows porous beaded titanium coupons p-NPP data normalised toDNA (Pico Green) with error bars of standard deviation. This data isfrom Table 3.

FIG. 20 shows polished titanium coupons p-NPP data normalised to DNA(Pico Green) with error bars of standard deviation. This data is fromTable 4.

Sample titanium alloy plates of various dimensions having surface areasranging from approximately 40 mm² to 100 mm² were washed cleaned anddried to form sample substrates. The prepared or sample substratesurfaces are approximately smooth. This can be seen most clearly fromFIG. 1 which is a view of the substrate surface taken by a ultra-highresolution scanning electron microscope.

A FEI Nova 200 NanoSEM ultra-high resolution Scanning ElectronMicroscope with a stated resolution of 1.8 nm at 3 kV and 1 nm at 15 kVusing immersion optics was used to characterise primary layers formed onthe titanium alloy substrate. The views or micrographs of the primarylayer show detail on the nanoscale. However, it will be appreciated thatother suitable methods and equipment may also be used to explore thesurface detail of the primary layer.

A surface of a prepared substrate sample was blasted with abrasivealumina particles otherwise known as alumina grit-blast. The process ofalumina grit-blasting roughens the surface of the substrate creating orpartially forming a primary layer which has a greater surface area thanthe surface area of the prepared substrate prior to grit-blasting. Thiscan be seen most clearly in FIGS. 2 and 5 a. The primary layer wascompleted by soaking the substrate with the partially formed primarylayer in a 4 molar solution of sodium hydroxide at 60° C. for two hours.FIG. 5b is a view of the completed primary layer which clearly shows thedevelopment of strut-like formations, fibres or fibrils of sodiumtitanate having dimensions on the nanoscale. The diameter or width ofthese fibrils fall within the range of between 1 and 20 nanometers.Approximately 80% of the fibrils have been measured as having a diameterin the range of 5 to 12 nanometers. The length of the fibrils arebetween 200 and 300 nanometers.

Five further samples of the prepared substrate were alumina grit blastedand soaked in sodium hydroxide solutions of concentration 3 molar, 4molar, 6 molar, 8 molar and 10 molar respectively at 60° C. for twohours and FIGS. 5c to 5g are views of the completed primary layer formedin each case. As can be seen from FIGS. 5c to 5g , the best etching,texturing or nanostructure formed or greatest density of fibrilformation was observed where the substrate was soaked in 4 molarsolution of sodium hydroxide. The greater the density of fibrilformation, the greater the surface area of the primary layer. Treatmentwith higher concentrations of sodium hydroxide was less effective andlead to re-dissolution of the nanostructure of the primary layerresulting in a smoother surface and thus a reduced surface area.

FIGS. 6a to 6c are views of a primary layer formed according to thepresent invention wherein the starting substrate and thus the initialtopography is different in each case. FIG. 6a is a view of a primarylayer which has been formed on a surface of a solid titanium alloysubstrate which has been blasted with abrasive alumina particles andsubsequently chemically treated by soaking in a 4 molar concentratedsolution of sodium hydroxide at 60° C. for 2 hours. FIG. 6b and FIG. 6care views of the primary layer which have been formed on a surface of aporous beaded titanium substrate and a titanium foam substraterespectively, which have been chemically treated in the same way as thesolid titanium alloy substrate.

The porous beaded titanium substrate and titanium foam substrate werenot subjected to any physical treatment such as in the case of the solidtitanium alloy substrate. It was found that the greater the surface areaof the starting substrate, the greater the surface area of the primarylayer formed. As can be quite clearly seen from FIGS. 6a to 6c thefibrils formed in the case of the titanium foam substrate, which had thegreatest starting substrate surface area, were the most fine, and thusfibril formation density was greatest presenting the highest primarylayer surface area. FIG. 9 is an SEM of a portion of a surface of aporous beaded titanium alloy prior to completing the primary layer.FIGS. 11a to 11d illustrate the development of the primary layer overtime when soaked in a 2 molar solution of sodium hydroxide at 60° C.

FIGS. 13a-13c are SEMs of increasing magnification (200, 500 and 1200times magnified respectively) of areas of the surface of TiAlNb alloyafter grit blasting with alumina particles but prior to soaking insodium hydroxide; the SEM employing a 2 kv beam to analyse the upperstructure of the completed primary layer. FIGS. 14a-14c are SEMs of thesame areas and at the same magnifications of the surface of the TiAlNballoy of FIGS. 13a-13c respectively after soaking in 4 molar sodiumhydroxide solution at 60° C. for 2 hours; the SEM employing a 2 kv beamto analyse the upper structure of the primary layer thus formed. FIGS.15a and 15b are SEMs of same areas and at the same magnifications of thesurface of the TiAlNb alloy of FIGS. 13b and 13c respectively; the SEMemploying a 15 kv beam to analyse the substructure of the primary layerformed.

The surfaces of the primary layers were analysed by scanning electronmicroscopy (SEM) before and after soaking the substrate in sodiumhydroxide solution to analyse surface topography and alumina contentthroughout the primary layer. This technique can be carried out atdifferent voltages which enables the surface and subsurface of theprimary layer to be analysed; the greater the voltage the deeper thepenetration of the beam. Titanium alloy has a higher average atomicnumber than alumina. The higher the average atomic number of thematerial being analysed using SEM, the greater will be the electronbackscatter and thus the brighter will be the SEM image.

Alumina has an average atomic number less than titanium alloy and thusan SEM image of titanium alloy with alumina present is darker thantitanium alloy without alumina. It is quite clear when comparing FIG. 1,which shows a titanium alloy substrate prior to alumina grit blasting,with FIGS. 13a to 13c , for example, which show the titanium alloysubstrate post alumina grit blasting, that quite a substantial amount ofalumina becomes embedded in the surface of the substrate forming theprimary layer. FIGS. 14a to 14c represent the primary layer of FIGS. 13ato 13c which have been completed by chemical treatment with sodiumhydroxide as described above. As can be seen, the SEM images of theprimary layer illustrated in FIGS. 14a to 14c are brighter than the SEMimages of the primary layer illustrated in FIGS. 13a to 13c because thesodium titanate layer formed masks the alumina particles present in theupper surface of the primary layer. Sodium titanate has an averageatomic number higher than that of alumina and thus the SEM image of thecompleted primary layer will appear brighter than the primary layercreated by alumina grit blasting and prior to treatment with sodiumhydroxide.

The higher voltage SEM images illustrate the composition of thesubsurface of the primary layer which is clearly darker and thus higherin alumina content than the upper surface regions. However, it is onlycritical to mask the alumina particles in the upper surface of theprimary layer which forms the implant-to-bone interface and thus is indirect contact with the bone, as contamination of the subsurface of theprimary layer with abrasive particles has little affect on the bondformed between the bone and implant.

Analysis of the reflectance of various substrates prior to and aftertreatment with 4 molar sodium hydroxide was also undertaken. As can beseen quite clearly from table I below, the greater the surface area ofthe primary layer, the less visible light is reflected. The titaniumfoam substrate which produced the primary layer having the greatestsurface area reflected only between 5 and 10% of the visible light. Allthe primary layers completed were black in colour when viewed by thenaked eye.

TABLE 1 4M NaOH 4M NaOH Control Control treated treated 4M NaOHWavelength Grit-Blast Porous Control Grit-Blast Porous treated (nm)Ti6Al4V Beaded Ti Foam Ti Ti6Al4V Beaded Ti Foam Ti 400 22.83 17.9917.61 12.71 4.78 5.56 410 23.23 18.32 17.86 12.61 4.94 5.71 420 23.618.58 18.05 12.53 5.06 5.82 430 23.95 18.75 18.19 12.47 5.12 5.88 44024.26 18.92 18.3 12.47 5.18 5.96 450 24.55 19.17 18.44 12.54 5.31 6.1460 24.82 19.42 18.6 12.67 5.45 6.25 470 25.11 19.6 18.8 12.88 5.59 6.39480 25.39 19.79 19.03 13.14 5.72 6.53 490 25.64 19.99 19.31 13.41 5.846.63 500 25.92 20.28 19.61 13.74 5.98 6.76 510 26.3 20.75 19.92 14.186.2 7 520 26.67 21.19 20.21 14.63 6.4 7.23 530 26.89 21.4 20.42 14.996.51 7.37 540 27.07 21.54 20.61 15.33 6.6 7.49 550 27.76 21.72 20.8 15.76.7 7.61 560 27.44 21.89 20.97 16.06 6.8 7.74 570 27.6 22.02 21.09 16.416.91 7.88 580 27.74 22.15 21.19 16.72 7 8.01 590 27.87 22.32 21.27 16.957.02 8.1 600 27.99 22.51 21.35 17.15 7.04 8.19 610 28.12 22.68 21.4817.36 7.15 8.31 620 28.28 22.85 21.67 17.6 7.3 8.46 630 28.52 23.0621.95 17.95 7.54 8.63 640 28.78 23.27 22.23 18.29 7.74 8.8 650 28.9 23.422.34 18.42 7.73 8.92 660 28.96 23.51 22.38 18.45 7.62 9 670 29.04 23.6522.41 18.46 7.55 9.06 680 29.14 23.79 22.46 18.47 7.52 9.11 690 29.3123.88 22.59 18.48 7.55 9.18 700 29.52 23.94 22.78 18.49 7.63 9.27

Sample titanium materials, titanium alloy coupons, having differentpre-treatments (grit blasted, polished, porous beaded) were compared forosteogenic activity on the surface after being chemically treated withan alkaline solution, compared to each other type and of pre-treatmentand to not being chemically treated.

The alkaline solution was a 4 molar solution of sodium hydroxide for 2hours at 60° (as described here before).

The pre-treatments of the titanium alloy coupons were polishing thesurface, grit blasting and porous beading as known in the art.

After the chemical treatment the titanium alloy coupons were insertedinto individual silicone tubes so that any fluid placed on to the couponremained on the test surface. Coupons were then sterilised.

Human mesenchymal stem cells were resurrected and passaged in suitablemedium and incubated overnight. After incubation the medium was replacedwith an osteogenic medium containing R-Glycerophosphate and this waschanged twice a week.

Live/dead staining on the cells was performed on all surface types atall time points.

The samples were subject to cell lysis and P-nitrophenol alkalinephosphatase p-NPP assay analysis to indicated osteogenic activity of thecells and thus, bone formation.

The results are as shown in Table 2 for grit blasted pre-treated couponsalkaline solution treated compared to not being subject to alkalinesolution.

TABLE 2 GB GB GB GB Non- GB Non- GB Non- GB Non- GB treated Alkalitreated Alkali treated Alkali treated Alkali Grit (Day (Day (Day (Day(Day (Day (Day (Day blasted 3) 3) 7) 7) 14) 14) 21) 21) p-NPP Rep 125.493 38.760 79.569 155.813 165.721 211.735 149.917 226.772 Rep 221.630 37.081 63.447 142.378 184.362 263.529 228.443 380.482 Rep 319.783 36.745 95.859 129.446 235.583 306.969 243.480 327.018 Pico Rep 16.702 5.965 5.227 7.439 11.863 7.439 10.388 9.651 Green Rep 2 6.7026.702 6.702 5.227 10.388 5.965 8.176 8.176 Rep 3 6.702 7.439 6.702 5.9659.651 7.439 10.388 8.176 Normal- Rep 1 3.804 6.498 15.222 20.945 13.97028.463 14.432 23.498 isation Rep 2 3.228 5.533 9.467 27.237 17.74844.183 27.940 46.535 Rep 3 2.952 4.939 14.304 21.703 24.411 41.26523.439 39.996 Mean 3.328 5.657 12.998 23.295 18.709 37.970 21.937 36.676Standard 0.435 0.787 3.092 3.435 5.286 8.362 6.878 11.872 deviation

Table 3 shows pre-treated porous beaded coupons with and withoutalkaline solution treatment.

TABLE 3 Porous Porous Porous Porous Non- Porous Alkali + Non- PorousAlkali + treated Alkali heat treated Alkali Heat Porous (Day (Day (Day(Day (Day (Day beaded 3) 3) 3) 7) 7) 7) p-NPP Rep 1 74.363 125.584150.271 76.404 295.273 198.369 Rep 2 75.874 161.187 187.553 153.259322.005 250.163 Rep 3 94.851 151.278 174.622 139.893 231.784 265.200Pico Rep 1 14.074 5.965 8.914 10.388 8.914 6.702 Green Rep 2 8.914 8.1768.914 11.125 9.651 6.702 Rep 3 8.176 8.176 8.176 9.651 7.439 6.702Normal- Rep 1 5.284 21.055 16.859 7.355 33.126 29.600 isation Rep 28.512 19.714 21.041 13.776 33.366 37.328 Rep 3 11.601 18.502 21.35714.495 31.158 39.572 Mean 8.466 19.757 19.752 11.875 32.550 35.500Standard 3.159 1.277 2.511 3.931 1.212 5.231 deviation Porous PorousPorous Porous Non- Porous Alkali + Non- Porous Alkali + treated AlkaliHeat treated Alkali Heat Porous (Day (Day (Day (Day (Day (Day beaded 14)14) 14) 21) 21) 21) p-NPP Rep 1 612.718 1137.336 734.683 636.108 744.708945.199 Rep 2 617.730 1132.324 709.622 843.282 968.589 1035.420 Rep 3729.671 1052.127 846.624 764.757 1100.579 1017.041 Pico Rep 1 11.1258.176 8.176 10.388 6.702 8.914 Green Rep 2 10.388 8.914 8.176 11.1258.914 9.651 Rep 3 11.125 8.176 9.651 10.388 8.176 9.651 Normal- Rep 155.074 139.102 89.855 61.235 111.121 106.041 isation Rep 2 59.466127.034 86.790 75.799 108.665 107.289 Rep 3 65.587 128.681 87.726 73.619134.606 105.384 Mean 60.042 131.606 88.124 70.218 118.131 106.238Standard 5.280 6.544 1.571 7.855 14.321 0.967 deviation

Table 4 shows polished coupons with and without alkaline solutiontreatment.

TABLE 4 Polished Polished Polished Polished Non- Polished Alkali + Non-Polished Alkali + treated Alkali heat treated Alkali heat (Day (Day (Day(Day (Day (Day Polished 3) 3) 3) 7) 7) 7) p-NPP Rep 1 20.287 37.92038.088 136.668 186.209 87.630 Rep 2 24.149 34.729 31.203 94.683 152.958118.866 Rep 3 20.119 36.913 31.203 67.477 148.927 132.133 Pico Rep 18.914 5.227 6.702 5.965 5.965 5.227 Green Rep 2 6.702 5.227 9.651 7.4395.227 5.227 Rep 3 12.600 4.490 5.965 22.921 5.227 5.227 Normal- Rep 12.276 7.254 5.683 22.913 31.220 16.764 isation Rep 2 3.603 6.644 3.23312.728 29.261 22.740 Rep 3 1.597 8.221 5.231 2.944 28.490 25.278 Mean2.492 7.373 4.716 17.821 29.657 21.594 Standard 1.021 0.795 1.304 7.2021.407 4.371 deviation Polished Polished Polished Polished Non- PolishedAlkali + Non- Polished Alkali + treated Alkali Heat treated Alkali Heat(Day (Day (Day (Day (Day (Day Polished 14) 14) 14) 21) 21) 21) p-NPP Rep1 206.723 527.509 158.271 288.590 −15.488 −20.500 Rep 2 265.200 470.703357.091 352.079 908.442 484.069 Rep 3 196.699 554.241 731.342 7.903527.509 388.836 Pico Rep 1 5.965 8.176 5.965 8.914 −0.671 0.067 GreenRep 2 6.702 5.227 5.227 8.176 8.176 9.651 Rep 3 7.439 5.965 7.439 0.0677.439 6.702 Normal- Rep 1 34.659 64.517 26.535 32.377 23.091 −308.186isation Rep 2 39.572 90.048 68.313 43.061 111.107 50.159 Rep 3 26.44192.923 98.311 118.804 70.911 58.020 Mean 33.557 82.496 64.387 37.71991.009 54.089 Standard 6.634 15.636 36.049 7.555 28.423 5.559 deviation

The results show that there is more Osteogenic activity where thecoupons have been chemically treated e.g. with an alkaline solutioncompared to not being chemically treated.

It will be appreciated that the primary layer may include variousbio-active materials including antimicrobials. It will also beappreciated that the primary layer may be further treated to impartanti-biofouling, cytogenic, catalytic, osteogenic or electrochemicalproperties to the implant.

It will be further appreciated that the substrate may comprise othermetals or alloys instead of titanium, for example, nitinol or zirconium.

It is envisaged that the material formed maybe subjected to furtherphysical treatment steps to improve or enhance the surfacecharacteristics of the primary surface layer. For example, on completionof the primary layer, the material can be rinsed in water or phosphatebuffered-saline solution to remove the alkali. After drying thematerial, it can be heated to a target temperature of between 300-600°C. The target temperature can be reached by raising the temperature ofthe material by 5° C. per minute. The target temperature, once reachedcan be maintained for at least one hour.

It will be appreciated that the invention is not limited to theembodiments hereinbefore described but may be varied in construction anddetail within the scope of the appended claims.

What is claimed is:
 1. A material suitable as an implant comprising atitanium or titanium alloy substrate comprising a first surface and aprimary layer on the first surface, the primary layer comprising aplurality of micron scale structures, wherein the plurality of micronscale structures collectively comprise a second surface, and a surfacelayer on the second surface, the surface layer comprising alkalititanates, wherein the thickness of the surface layer is between 100 and500 nm, wherein the surface layer comprises a plurality of nanoscalestructures, and wherein the plurality of nanoscale structurescollectively comprise a third surface.
 2. The material of claim 1,wherein the alkali titanates comprise sodium titanate.
 3. The materialof claim 1, wherein the alkali titanates comprise discrete elements orfibrils comprising a width in the range of 2 to 20 nm and a length inthe range of 200 nm to 300 nm.
 4. The material of claim 1, wherein thethickness of the surface layer is between 100 and 500 nm.
 5. Thematerial of claim 1, wherein the primary layer further compriseshydroxyapatite.
 6. The material of claim 1, wherein the primary layerfurther comprises titanium oxide.
 7. The material of claim 1, whereinthe first surface has a first surface area, the second surface has asecond surface area, and the third surface has a third surface area,wherein the third surface area is greater than the second surface area,and wherein the second surface area is greater than the first surfacearea.
 8. The material of claim 7, wherein the third surface area isbetween 1000 and 50000 times greater than the first surface area.
 9. Thematerial of claim 1, wherein the third surface comprises a reflectanceto visible light in the range of 1% to 20%.
 10. The material of claim 9,wherein the third surface comprises a reflectance to visible light inthe range of 6% to 10%.
 11. The material of claim 1, wherein the primarylayer further comprises alumina in a concentration greater aconcentration of alumina in the substrate.
 12. The material of claim 11,wherein the surface layer of the primary layer comprises alumina in aconcentration lesser than a concentration of alumina in a subsurface ofthe primary layer.
 13. The material of claim 1, wherein the primarylayer is formed by a process comprising soaking at least a portion ofthe substrate in an alkaline solution comprising a concentration of 2 to6 molar at a temperature of 50° C. to 70° C., for 1 to 24 hours.
 14. Amaterial suitable as an implant comprising a titanium or titanium alloysubstrate comprising a first surface and a primary layer on the firstsurface, the primary layer comprising a plurality of micron scalestructures, wherein the plurality of micron scale structurescollectively comprise a second surface; a surface layer on the secondsurface, wherein the surface layer comprises alkali titanates, whereinthe alkali titanates comprise a plurality of nanoscale structures, andwherein the plurality of nanoscale structures collectively comprise athird surface; and alumina in a concentration greater than aconcentration of alumina in the titanium or titanium alloy substrate.15. The material of claim 14, wherein the surface layer of the primarylayer comprises alumina in a concentration lesser than a concentrationof alumina in a subsurface of the primary layer.
 16. The material ofclaim 14, wherein the alkali titanates comprise discrete elements orfibrils comprising a width in the range of 2 to 20 nm and a length inthe range of 200 nm to 300 nm.
 17. The material of claim 14, wherein theprimary layer further comprises hydroxyapatite.
 18. The material ofclaim 14, wherein the primary layer further comprises titanium oxide.19. The material of claim 14, wherein the first surface has a firstsurface area, the second surface has a second surface area, and thethird surface has a third surface area, wherein the third surface areais between 1000 and 50000 times greater than the first surface area. 20.The material of claim 14, wherein the third surface comprises areflectance to visible light in the range of 1% to 20%.